Molecular Constructs for Differentiating a Target Molecule from an Off-Target Molecule

ABSTRACT

Molecular constructs, populations thereof, arrays, compositions, methods and kits for differentiating a target molecule from an off-target molecule are provided.

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 61/483,323, filed May 6, 2011. Theforegoing application is incorporated by reference herein.

This invention was made with government support under Grant No.R43-AI074089 awarded by the National Institutes of Health/NationalCancer Institute and Grant No. R43-GM078946 awarded by NationalInstitutes of Health/National Cancer Institute. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to differentiating a target molecule froman off-target molecule, the target molecule having a target domain andthe off-target molecule having an off-target domain that differs fromsaid target domain.

SUMMARY OF THE INVENTION

In accordance with the present invention, a molecular construct (orcomplex) for differentiating a target molecule from an off-targetmolecule is provided in which the target molecule has a target domainand the off-target molecule has an off-target domain that differs fromsaid target domain. Such a molecular construct comprises

a. a first domain that is capable of binding to said target domain, and

b. a second domain that is at least partially hybridizable with saidfirst domain, wherein said first domain is capable of binding to saidoff-target domain, wherein a hybrid of said first domain with saidsecond domain is

-   -   i. in a state of predetermined stable equilibrium in the absence        of said target domain and    -   ii. in a state of predetermined metastable equilibrium in the        presence of said target domain; and        wherein the free energy of displacement of said second domain by        said target domain from said hybrid of said first domain with        said second domain is energetically more favored than the free        energy of displacement of said second domain by said off-target        domain from said hybrid of said first domain with said second        domain.

In the molecular construct of the present invention, the off-targetdomain may also be generally homologous with respect to the targetdomain. Further, in the molecular construct of the present invention,the off-target domain may also differ from the target domain eitherchemically by at least one functional group or conformationally or both.Further, in accordance with the present invention, a population of suchmolecular constructs (or probe complexes) may be provided. Such apopulation may comprise

a. a first subpopulation of at least one said molecular construct withrespect to a first target domain and

b. a second subpopulation of at least one said molecular construct withrespect to a second target domain, wherein said first target domaindiffers from said second target domain. In another embodiment, thepopulation comprises

a. a first subpopulation comprising at least one said molecularconstruct with respect to a first off-target domain and

b. a second subpopulation comprising at least one said molecularconstruct with respect to a second off-target domain, wherein said firstoff-target domain differs from said second off-target domain.

In accordance with another aspect of the instant invention, arrays areprovided. In a particular embodiment, the arrays comprise at least onebiosensor element on a solid support. Each biosensor element maycomprise at least one molecular construct (or probe complex) of theinstant invention. In a particular embodiment, the array comprises aplurality of populations of molecular constructs of the instantinvention for 1) detecting a plurality of different target molecules ina sample and/or 2) determining the relative number of target moleculesin a sample, wherein the free energy of displacement of each of thosepopulations differs with respect to each other of those populations soas to be capable of 1) identifying different target molecules in thesample and/or 2) determining the relative number of target molecules ofa population from another population in said sample. In a particularembodiment, the array comprises a plurality of populations of molecularconstructs of the instant invention for identifying a plurality ofdifferent target molecules in a sample and determining the relativenumber of each of those different target molecules in the sample,wherein the free energy of displacement of each of those populationsdiffers with respect to each other of those populations so as to becapable of identifying each different target molecule in the sample andthe free energy of displacement of each of those populations differswith respect to each other of those populations so as to be capable ofdetermining the relative number of each of those different targetmolecules of a population from another population in the sample. In aparticular embodiment, the number of different targets is from about 2to 1000 or more, about 2 to about 10, about 2 to about 100, about 100 toabout 1000, or greater than 1000. In a particular embodiment, the ratioof the free energy of displacement of one population to anotherpopulation is about 1.1, greater than about 1.1, about 1.1 to about 1.2,1.5, or 2.0, about 1.2 to about 1.5, about 1.5 to about 2.0, or greaterthan 2.0. Kits comprising the arrays of the instant invention are alsoprovided.

In accordance with another aspect of the instant invention, methods fordifferentiating a target molecule from an off-target molecule areprovided. The method may be used to detect and/or quantitate a nucleicacid sequence of interest or target molecule. In a particularembodiment, the method comprises contacting at least one molecularconstruct (or probe complex) of the instant invention with a sample(e.g., a biological sample). In a particular embodiment, the methodcomprises a) contacting a population of probe complexes (or moleculeconstructs) with a sample, and b) detecting the formation of complexesbetween the probe strand and a nucleic acid molecule from the sample,wherein the presence of such complexes is indicative of the presence andor quantity of the nucleic acid sequence of interest. The population ofprobe complexes may comprise at least i) a first subpopulation of probecomplexes comprising a probe strand and a competitor strand, wherein thecompetitor strand comprises at least one hybridization domain(particularly at least two) and at least one domain which does nothybridize with the probe strand, wherein the hybridization domain is atleast partially complementary to the probe strand, and ii) a secondsubpopulation of probe complexes comprising a probe strand and acompetitor strand, wherein the competitor strand comprises at least onehybridization domain (particularly at least two) and at least one domainwhich does not hybridize with the probe strand, wherein thehybridization domain is at least partially complementary of the probestrand, wherein the non-hybridization domain of the second subpopulationdiffers from the non-hybridization domain of the first subpopulation. Ina particular embodiment, the probe and competitor strands are reversed.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A provides a schematic of the principle of action of QuantitativeRecognition Arrays (DNA-meter and Apta-meter). FIG. 1B provides aschematic of the action of competitive surface probes. Strands P (probe)and C (competitor) are attached via neighboring groups on the surface. Cis partially complementary to P. The target T binds P and displaces C.(I) T is a nucleic acid strand complementary to P. (II) T is a proteintarget to the aptamer P. In a particular embodiment of the instantinvention, the P and C labeling may be reversed in the probe complex.FIG. 1C also provides a schematic of an embodiment of the instantinvention.

FIGS. 2A-2D provide images of results obtained from the interactions ofcompetitive surface probes with Cy3 labeled targets. Targetconcentrations are as illustrated under each figure.

FIG. 3 provides an illustration of how the DNA meter can be used todistinguish between single nucleotide polymorphisms (SNPs). For thewild-type, a characteristic pattern of competitive surface probes (CSPs)turned “on” is observed. When a SNP occurs, the pattern of probes turned“on” helps to define which SNP is prevalent.

FIGS. 4A-4C provide simulations of the target binding behavior ofcompetitive surface probes. FIG. 4A shows 40° C. plots of the fractionof bound probe vs. total target concentration for target in excesscompared to probe. FIG. 4B provides 40° C. plots for probe concentrationfixed at 10 nM. Inset is a plot of critical concentration for ½saturation vs. K₁, with data on a log scale. FIG. 4C shows curves atvarious temperatures for the coil probe, under conditions of excesstarget.

FIGS. 5A and 5B provides simulations of PCR amplification of mecA targetin the presence of mecA competitive surface probe. All calculations arefor an annealing temperature of 50° C., and for a starting targetconcentration of 1×10⁻¹⁵M. FIG. 5A is a simulation under typicalsolution conditions with 0.1 μM MecA probe, 100 mM KCl, 1 mM MgCl₂. FIG.5B provides a simulation under mimicking those on a surface, where theprobe concentration is negligible compared to the target. For FIG. 5A,half-saturation of probe occurs at 5×10⁻⁸ M target, corresponding to acritical cycle number of 26. For FIG. 5B, half-saturation occurs whenthe concentrations of the perfect hybrid and the single mismatch are8×10⁻¹² and 4×10⁻¹⁰ M, respectively, corresponding to critical cyclenumbers of 13 and 19.

FIG. 6 provides a schematic of one manifestation of the Competitor●Probe(C*●P) complex for the DNA meter in which the competitor probe complexis destabilized incrementally and in a predictable manner by increasingthe size of a “recognition-neutral” T bulge loop in the centre of thecompetitor strand. Besides all-T bulge loops of varying size, numerousother modifications can be incorporated into the competitor strand topredictably modulate the stability of the “target recognition neutral”interaction between the competitor “masking tape” and the probe strand.The nucleotide sequences are SEQ ID NO: 11 (competitor strand) and SEQID NO: 12.

FIG. 7A provides normalized optical melting curves showing how a changein the “recognition neutral” T-bulge in the competitor strand modulatesthe thermal stability of the Probe●Competitor complexes (C*●P). FIG. 7Bprovides normalized optical melting curves that result when equimolaramounts of Target (T) strand are added to the Probe●Competitor complexes(C*●P) in FIG. 7A. The strand displacement and exchange (C*●P and T toP●T and C) is seen in the UV melts wherein increasing the loop size inthe competitor strand results in destabilized Probe●Competitor complexesand a lower temperature at which strand exchange occurs.

FIG. 8A shows UV melting curves and FIG. 8B provides the correspondingdifferential scanning calorimetry (DSC) melting curves of an equimolarmixture of T2●P, T4●P, and T8●P competitor●probe complexes in which thethermal stability of the (C*●P) is systematically modulated by thepresence of “recognition neutral” T-bulges of increasing size (T2, T4,T8) upon addition of increasing amounts of Target strand T.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the instant invention, energetically tunable probesare provided. Watson-Crick, canonical G●C and A●T pairing interactionsrepresent the molecular language/code by which two complementary DNAsequence domains selectively recognize and bind/hybridize to oneanother. This selective recognition pattern has been employed to designspecific DNA sequences which can probe for the presence of complementarytarget DNA domains. Applications of such probe-target hybridizationassays have been extensive; including testing (probing) for the presenceof a particular target gene, gene transcript, secondary structuralelement, or functional/regulatory sequence domain; creating a primingduplex domain for PCR or sequencing methods; modulating the activity ofa nucleic acid construct; etc. More recently, probe-target hybridizationassays, including variants, as elaborated below, have been used to: mapactively transcribed genes in entire cell lines (the so calledtranscriptome), as well as in different tissue types, in systems biologyapplications, and to identify genetic changes in cancerous tissues.

Simple hybridization between single stranded probes and single strandedtargets often lacks the requisite selectivity, with secondary andtertiary probe-target “hits” and “misses” compromising manyapplications. Accordingly, greater stringency is required than what hasbeen afforded by simple hybridization. This realization led to thedesign of a number of hybridization assays predicated on strandinvasion, strand displacement, or strand exchange strategies in whicheither the probe or the target, or both, were in a duplex or otherpre-organized state. For example, the probe may be in the duplex stateand a successful target hit requires strand displacement. Using such anapproach, one could directly detect subtle differential stabilitiesbetween one target and another, rather than having to distinguish smalldifferences between two large numbers derived from independentmeasurements. While an improvement over traditional single strandedprobe/single stranded target assays, such direct differential approachesstill required subtle tuning of the relative stabilities of probes andtargets to achieve the stringency required in many applications. To thisend, efforts focused on designing into the probe specific mismatches,modified bases, and even non-nucleoside components to achieve greaterselectivity for the assay such that the duplex probe comprises amismatch or non-Watson-Crick imperfection. However, such “tuning” of thedifferential energetics often also tampered with the underlyingWatson-Crick recognition code, thereby adding an additional layer ofcomplexity and, at times, ambiguity, to the resulting interpretation ofthe “hit” (successful hybridization). As a result, even these secondgeneration hybridization assays frequently exhibited unacceptable levelsof false positives and false negatives.

Specificity is defined as the difference (ΔΔG) of the binding freeenergy (ΔG) of probe to a specific target and probe binding to anoff-target. Stringency can then be defined as the relative contributionof binding selectivity to the overall binding free energy of probe totarget. Stringent conditions are those where binding to the specifictarget is stable, but where binding to the non-specific target is not.Seen in this light, it is clear that if affinity can be attenuated whilemaintaining constant selectivity, then stringency will necessarily beenhanced and can be optimized. Selectivity refers to the ability of theprobe to distinguish and preferentially recognize/bind a specific targetfrom among an ensemble of (closely related) off-targets. Selectivitymay, in looser terms, also be considered as the ability of a ligand tointeract with a particular population of target molecules in preferenceto others. The term selectivity may in some cases be used to imply adegree of specificity. In the limit of one off-target, the definition ofspecificity and selectivity are identical.

With duplex hybridization, probe binding to non-specific targetdiffering by a single mismatch compared to a specific target iscorrelated with a free energy difference of only a few kcal per mole,roughly independent of salt and temperature. Under high salt and lowtemperature conditions, the hybridization of two strands to form aduplex is so strongly favored, that mismatches are often well tolerated,and the ability to discriminate between specific and non-specifictargets will be compromised. In order to discriminate between mismatchand a match target directly it is better to work under low salt and/orhigher temperature conditions where the overall hybridization freeenergy is reduced and stringency is enhanced. Under these circumstancesthe difference of a few kcal per mole between probe binding to specificand non-specific targets becomes significant compared to the total freeenergy of hybridization, and can be readily measured. Under conditionswhere melting occurs, the overall hybridization free energy approacheszero, thereby enhancing the ability to monitor differences in freeenergy due to mismatches or other defects.

Temperature is a very useful and well recognized parameter for enhancingstringency (i.e. the contribution of binding selectivity to the overallprobe-target binding free energy). Less well appreciated, but likewiseuseful, is the concept of modulating binding affinity (e.g., strength ofan interaction between two entities, Ka) through competitiveinteractions.

One very useful actualization of this principle is found in competitiveprobe technology (Gelfand et al. (1999) Proc. Natl. Acad. Sci.,96:6113-8; Plum et al. (2001) Biopolymers 61:214-23; U.S. Pat. No.6,815,163). Competitive probe technology allows the measurements ofsmall differences in free energy between the hybridization of twodifferent target molecules to the same probe molecule. The referenceduplex AD is formed from a fluorescent acceptor labeled probe A and adonor labeled target D. Competition with an unlabeled strand X resultsin the formation of competitor/acceptor duplex and a decrease in FRET.Such competitive constructs are capable of distinguishing free energydifferences as small as 1 kcal per mol, and can thereby be used todistinguish SNPs and a variety of small-scale defects, even in thepresence of a very stable duplex. Another significant advantage ofcompetitive probes is that they can be designed and optimized to avoidkinetic traps that are a significant concern for molecular beacons andother hairpin probes, particularly at lower temperatures (Braunlin etal. (2004) Biopolymers 74:221-31).

It is shown herein that one can selectively tune the energetics of aduplex or pre-engaged probe without altering the canonical Watson-Crickrecognition code of the component probe strand used to seek out(hybridize to) a complementary target domain. Using this strategy, oneis able to enhance stringency without compromising targetidentification. Here it is demonstrated that this goal can be achievedin one manifestation by initially complexing or “tying up” a probestrand with a complementary competitor strand (also referred tometaphorically here as a “masking tape” strand) in which the competitorstrand element used to modulate the stability of the overall probecomplex (or molecular construct) (and therefore the “availability” ofthe probe strand to bind target) is extra-helical and does not alter theWatson-Crick recognition interface of the probe. In a particularembodiment, the strength (stability) of the resulting probe complex (ormolecular construct) is modulated by changing the size and/or sequenceof a non-hybridizing domain, such as a bulge or loop, within thenon-probing, “masking tape” strand. As such, there is no requirement forone to alter the Watson-Crick recognition code of the probe strand. Thesignificant feature in such constructs is that one is able to tune theenergetics of the probe-competitor complex, and therefore theavailability of the probe strand to hybridize to its complement target,as a function of target concentration, without altering the recognitionelements of the probe for the target. This represents a qualitative andquantitative advance relative to duplex probe approaches which requireless refined and predictable tuning via alterations in probe componentsessential for selective target recognition.

Using such molecular constructs (complexes), it is shown herein that theprobe strand in complex with the most destabilizing masking tape strandis selectively displaced from the probe complex at low target strandconcentration to form the probe-target complex that defines a successfulhit. Only at sequentially higher target concentrations are the morestable masking tape strands displaced from the probe, in precisely theorder that maps with the relative stabilities of the initial probecomplexes. Such bulge or loop families of energetically discrete probecomplexes may be referred to as “tuning fork” probes. It is demonstratedthat such tunable probe complexes not only can selectively detect thepresence of target sequences, but also can quantitate the amount oftarget present. This aggregate capacity allows for the referral to themethodology described here as a DNA meter. However, the instantinvention is not limited to DNA. Indeed, the competitor-probe complexand target-probe complex may be complexes of any nucleic acid molecules(e.g., DNA, RNA, and nucleic acid analogs or mimics (inclusive of lockednucleic acids, PNAs, and other base or backbone modifications)) or otherspecific binding pair including proteins and polypeptides.

For simplicity, the following description generally details the use ofnucleic acid molecules. However, as stated above, the instant inventionencompasses the use of any specific binding pair.

According to one aspect of the instant invention, the complexes comprisenucleic acid molecules. In a particular embodiment, the competitorstrand comprises at least one, particularly two or more regions, whichspecifically hybridize with the probe strand and one or more otherregions (e.g., a bulge or loop region) which do not hybridize with theprobe strand. In a particular embodiment, the competitor strand maycomprise a single non-hybridizing domain between two hybridizing domainsor the competitor strand may comprise two non-hybridizing domainsinterspersed among three hybridizing domains, etc.

The non-hybridizing domain(s) may be of any length. In a particularembodiment, the non-hybridizing (e.g., loop) domain comprises from 1 toabout 50 nucleotides, particularly from 1 to about 25 nucleotides. Thesequence of the non-hybridizing domain does not specifically hybridizewith the probe, particularly under the assay conditions employed. Forexample, the sequence of the non-hybridizing domain may have less than50% identity, less than 40% identity, less than 30% identity, or lessthan 25% identity with the probe sequence. In a particular embodiment,the nucleotides of the non-hybridizing domain are the same (e.g., allthymidines). Alternatively, the sequence of the non-hybridizing domainis variable.

The domains which specifically hybridize with the probe strand may be atleast complementary, particularly completely complementary, to the probestrand and/or target strand. In other words, the sequence of the two ormore hybridizing to domains when considered in tandem as a singlesequence create a sequence that is complementary, particularlycompletely complementary, to the probe and/or target strand. Thehybridizing domain(s) may be of any length. In a particular embodiment,the hybridizing domain comprises from 1 to about 50 nucleotides, from 1to about 25 nucleotides, or from about 10 to about 25 nucleotides.Notably, the competitor strand need not be completely complementary forthe entire length of the probe strand (or target strand). Indeed, theprobe, competitor and target need not be the same length (and typicallywill not be).

In a particular embodiment, a molecular construct with a singlehybridizable domain (at least partially complementary) and a singlenon-hybridizable domain effect for modulating binding to the probestrand might be formed with these domains at least partially overlappingin sequence space and the non-hybridizable domain forming a secondarystructure on its own, i.e, for example, a probe that contains a numberof cytosines at either 5′ or 3′ terminus, in which case the hybridizablepart of the competitor strand contains complimentary guanines. If thenon-hybridizable domain is also rich in guanines, these could fold uptogether with the guanines in the hybridizable domain to form a Gtetraplex in competition to binding to the probe strand, therebymodulating the interactions with the probe strand. In other words—thenon-hybridizable domain forms a self structure with part of thehybridizable domain in competition to binding to the probe strand (andthat would almost make it a nested arrangement). In a particularembodiment, the hybridizable domain and the non-hybridizable domain donot necessarily correspond to clearly distinct regions in (linear)sequence space, but they could partially overlap.

The instant invention also encompasses populations of probe-competitorcomplexes. The population of probe-competitor complexes may comprise atleast one subpopulation. In a particular embodiment, each subpopulationdiffers from other subpopulations by having a different non-hybridizingdomain. For example, a population of the instant invention may comprisea first subpopulation having a non-hybridizing domain comprising a loopof X nucleotides, a second subpopulation having a non-hybridizing domaincomprising a loop of X+Y (e.g., 1) nucleotides, and so on. In aparticular embodiment, the population comprises a “ladder” ofsubpopulations wherein the length of the loop domain is incrementallyincreased. The population of probe-competitor complexes may be insolution or spatially confined (e.g., attached to a solid support). Whenattached to a solid support, the subpopulations may be attached in anordered or array format to the solid support.

In accordance with the present invention, a molecular construct fordifferentiating a target molecule from an off-target molecule may beprovided in which the target molecule has a target domain and theoff-target molecule has an off-target domain that differs from saidtarget domain. Such a molecular construct comprises

a. a first domain that is capable of binding to said target domain, and

b. a second domain that is at least partially hybridizable with saidfirst domain, wherein said first domain is capable of binding to saidoff-target domain; wherein a hybrid of said first domain with saidsecond domain is

-   -   i. in a state of predetermined stable equilibrium in the absence        of said target domain and    -   ii. in a state of predetermined metastable equilibrium in the        presence of said target domain; and wherein    -   i. the free energy of displacement of said second domain by said        target domain from said hybrid of said first domain with said        second domain is energetically more favored than    -   ii. the free energy of displacement of said second domain by        said off-target domain from said hybrid of said first domain        with said second domain.

In the molecular construct of the present invention, the off-targetdomain may also be generally homologous with respect to the targetdomain. Further, in the molecular construct of the present invention,the off-target domain may also differ from the target domain eitherchemically by at least one functional group or conformationally or both.In a particular embodiment, the first and second domains may be on thesame molecule or different molecules.

Further, in accordance with the present invention, a population of suchmolecular constructs may be provided. Such a population may comprise

a. a first subpopulation of at least one said molecular construct withrespect to a first target domain and

b. a second subpopulation of at least one said molecular construct withrespect to a second target domain, wherein said first target domaindiffers from said second target domain.

In a particular embodiment, the population comprises

a. a first subpopulation comprising at least one said molecularconstruct with respect to a first off-target domain and

b. a second subpopulation comprising at least one said molecularconstruct with respect to a second off-target domain, wherein said firstoff-target domain differs from said second off-target domain.

As explained hereinabove, molecular constructs and populations thereofmay be in solution (spatially unconstrained) or may be spatiallyconstrained, such as in competitive surface probes (hereinafter “CSPs”;e.g., competitor-probe complexes linked to a solid support) as are morefully described hereinafter. Spatially constrained populations andsubpopulations thereof are may be presented in accordance with thepresent invention as arrays.

It should be noted that the term “differentiating” may includedetection, identification, quantification, surveillance, diagnosis,genotyping, profiling, fingerprinting, isolating, and/or a combinationthereof of a target molecule from an off-target molecule, and evenseparation and/or purification.

In accordance with the instant invention, arrays for differentiating,such as detecting and quantitating, target molecules are provided. In aparticular embodiment, the array comprises more than one biosensorelement on a solid support, wherein each biosensor element comprises aspecific binding pair (e.g., comprising a probe and a competitor) linkedto the solid support. Each member of the specific binding pair mayindividually be a nucleic acid, peptide nucleic acid, or otherhybridizable entity. The arrays of the instant invention may comprisemore than one series (subset) of biosensor elements (e.g., about 2 toabout 100 (or more), about 2 to about 20, about 2 to about 10, or about5 to about 10 biosensor elements). Each series of biosensor elements maybe designed towards a specific or different target molecule. In aparticular embodiment, each biosensor element of a series comprises thesame probe (and/or competitor) in the specific binding pair of eachbiosensor element of the series. In still another embodiment, eachseries of biosensor elements comprise a different competitor (or probe)in the specific binding pair of each biosensor element of the series,wherein the different competitors have different binding affinities forthe probe (e.g., by having different or more mismatches).

In accordance with the instant invention, kits comprising at least onearray of the instant invention are also provided. The kits may furthercontain buffers.

According to another aspect of the instant invention, methods ofdetecting the presence of a target molecule and/or quantitating theamount of a target molecule in a sample are also provided. The methodscomprise contacting the sample with the competitor-probe complexes(e.g., an array) of the instant invention and detecting/monitoring thepresence of the target molecule or molecules. The method may comprisewashing unbound molecules from the array, but does not require washsteps. The method may also comprise a denaturing step such as a heatingstep to disrupt the specific binding pairs on the array, optionally,while cooling in the presence of the target molecule sample. The methodmay also comprise a step involving solution or solvent changes in orderto disrupt specific binding pairs (e.g., changes in temperature (e.g.,heating), changing the pH and/or adding a denaturant (e.g., urea,guanidine hydrochloride, and the like). In particular embodiments, thetarget molecule is detectably labeled (e.g., with an isotope,radioisotope, fluorescent compound, etc.). In other embodiments thetarget molecule is not labeled. In yet another embodiment, the targetmolecule for the surface probes may be part of a solution probe forbinding a solution target. In this manner a labeled solution probemolecule can be released from complex with an unlabeled target insolution and thereby provide a labeled molecular entity for binding tothe surface probes.

False positives in research and diagnostics represent a significantlimitation of increasingly sensitive biosensor technologies. Attempts toovercome false positives by reducing sensitivity will lead to anincrease the likelihood of false negatives. The technology of thepresent invention provides an approach to overcome this trade-offbetween false-positives and false-negatives. Molecular components of thepresent invention are exemplified and provided by Competitive SurfaceProbes (CSPs). The integration of CSPs into Quantitative RecognitionArrays (e.g. DNA-meters and Apta-meters) provides means to a) quantifytarget concentrations and b) substantially reduce the occurrence offalse-positives in research and diagnostic assays. By carefully matchingthe competing thermodynamics of self-hybridization and target binding,CSPs allow target binding specificity to be maintained, while bindingaffinity is attenuated over a range useful for biotechnological anddiagnostic applications. For the DNA-meter, the targets are specificnucleic acid molecules. For the Apta-meter the targets are proteins,small-molecules, or other molecular entities for which nucleic acidaptamers have been discovered. In either case, the QuantitativeRecognition Array provides a series of digital switches that define thetarget identity and concentration by the types and numbers of switchesof each type that are turned “on”. The experimental results describedherein may utilize fluorescence detection technologies as outputsignaling means. However, the fundamental features of CSPs andQuantitative Recognition Arrays are independent of platform, and arethus applicable to a wide variety of biosensor technologies.

The conceptual basis of the competitive surface probes (CSP) technologyof the present invention derives in part from solution competitiveprobes (Gelfand et al. (1999) Proc. Natl. Acad. Sci., 96:6113-8; Plum etal. (2001) Biopolymers 61:214-23), from the nucleic acid switches forprotein sensing and screening (DeCiantis et al. (2007) Biochem.,46:9164-73), from tunable affinity ligand and bimolecular probesconcepts, and from studies of DNA triplex stability (Roberts et al.(1991) Proc. Natl. Acad. Sci., 88:9397-401). The integration of CSPsinto quantitative biorecognition arrays represents a cross-platformmolecular technology of the present invention empowering the developmentof biosensors.

A Quantitative Recognition Array of the present invention may comprise aseries of molecular construct biosensor elements, with each elementcoated with one of a series of Competitive Surface Probes (CSPs). EachCSP will turn “on” if its target concentration exceeds a certainthreshold. Target identities and concentrations are thus defined by theunique sequence of CSPs that are turned “on”. As described below, eventhough individual CSPs may show cross-reactivity under some targetconcentrations, the pattern of CSP switching can be used to uniquelydefine target identities and concentrations over a wide range of totaltarget concentration. Hence, the incorporation of arrayed CSPs (e.g.,DNA-meters or Apta-meters) onto biosensor surfaces provides a powerfuland widely applicable solution to the problems of false-positives and oflow-cost quantification for a wide range of research and diagnosticapplications.

If the CSP-coated elements for a single nucleic acid target are arrangedleft to right from lower to higher threshold, then a model DNA Meterwill manifest the behavior shown in FIG. 1A. If the CSP-coated elementsare based on aptamers for a single target, then FIG. 1A can equally wellrepresent a model Apta-meter. Key features of CSPs are illustrated inFIG. 1B. These constructs comprise two strands that are attached to asurface within a hybridizable distance of each other. The probe strand Pbinds the target and the competitor strand C is designed to hybridizewith a range of affinities to P. Depending on the context, the C strandmay have environmentally sensitive switch elements.

In its most basic form, under a defined set of solution conditions, boththe DNA-meter and the Apta-meter provide a digital readout of targetidentity and concentration based on which of a series of engineeredprobes are turned on and which remain off. Since hundreds of individualbiosensors can be arrayed on a surface, such as a coverslip, multipledigital readouts can be obtained for multiple targets, allowing for theprecise determination of scores of individual target concentrations. Forthe DNA-meter, when combined with initial rapid pre-amplification steps,e.g. by end-point PCR or isothermal amplification methods, thetechnology of the present invention represents a highly attractive andcost-effective alternative to qPCR for diagnostic applications. For theDNA-meter, by utilizing multiple probes per target and multiple targetsper amplicon, the presence of genetic anomalies is readily detected by adecrease in the number of probes turned on for the mutated targetcompared to other targets on the same amplicon. Similarly, for theApta-meter, closely related target proteins can be discriminated, e.g.,proteins that differ by the extent or nature of post-translationalmodification.

In contrast to traditional microarray measurements, for which slides aredried prior to imaging, for Quantitative Recognition Arrays,measurements may be made for arrays that are immersed in targetsolution.

CSPs modified with 5′ and 3′ amino groups may be attached as functionalbimolecular probes to dextran coated slides (see, e.g.,PCT/US2006/047523). Because CSPs are bound to dextran polymers, theirbehavior is significantly more “solution-like” than for typical arraysthat are coated in a two-dimensional manner on the top of a glass slide.Dextran polymers may be replaced by other spacer groups such as oligo-Tregions and PEG spacers. The instant approach allows for the focus onspecific interactions rather than on artifacts relating to surfaceeffects.

Briefly, dextran may be covalently coupled to epoxy-silanated slides andsubsequently activated with NaIO₄ to create pairs of reactive aldehydesby partial oxidation of some of the carbohydrate monomers atcis-hydroxyl positions. Typically, gels are dried to ˜50% humidity priorto spotting. In other embodiments, arrays of CSPs may be prepared on theslides as follows: First, C6 amino CSPs may be dissolved in 50 mM NaHCO₃pH 8.5 with 100 mM KCl and 4 mM MgCl₂ to a concentration of between 1-10μM. Just before spotting, NaCNBH₃ is added to a concentration of 100 mM.The dried gel is spotted and allowed to sit at room temperature in 50%humidity overnight. Then, the slide is treated with 50 ml of 1 mg/nilNaBH4 for 30 minutes at room temperature to deactivate any remainingaldehyde groups, washed with several changes of water, 20% propanol, andfinally hybridization buffer before incubation with the target.

CSPs provide many advantages. For example, similar targets can bedistinguished over a wide range of concentrations with CPSs. Forsolution probes, the concentration of probe-target complex must be inthe nanomolar range or higher in order for a significant fluorescencesignal to be monitored. However, if the probe-target complexconcentration is in the nanomolar range, then the total concentrationsof both probe and target must be at least in this range as well. If theequilibrium dissociation constant for the formation of probe-targetcomplex is sub-nanomolar, then the binding of target to probe will beessentially quantitative, and binding curves, as monitored byfluorescence, will not depend significantly on the details of the probeutilized. Moreover, small differences between targets, will beimpossible to discern based on equilibrium curves alone. In starkcontrast, for CSPs the concentration of target will typically be insignificant excess over that of surface-bound probe, and the fraction ofprobe bound will be defined by a) the equilibrium constant forprobe-target association and b) the concentration of free target insolution. Hence, in this case, the observed signal will dependsignificantly on both the nature of the probe and target molecules andon the free concentration of target.

In addition to the above, CSPs provide a high level of multiplexingcapacity. Individual sensor elements can be derivatized with differentsurface probes in order to obtain a signature of the type and nature oftargets in solution. Discrimination is based on the position ofindividual probes within the surface array and does not require thesimultaneous monitoring of multiple fluorophores. One useful aspect ofthis multiplexing capability is as follows: consider a DNA meter withmultiple competitive surface probes for each target on a particularamplicon and with multiple targets per amplicon. The DNA meter willrespond in a characteristic pattern of “on” signals to increasingamplicon concentration. However, this pattern will be perturbed if oneof the target sequences on the amplicon has a SNP. In this case, all ofthe probes for that particular target will turn on at higherconcentrations than anticipated, compared to the probes for the othertargets on that amplicon. Consequently, the multiplexing capability ofthe DNA meter will allow the accurate and sensitive detection of SNPs.The principle behind this application of CSPs to determine SNPs andother genetic abnormalities is shown in FIG. 3.

Similarly, for the Apta-meter, closely related proteins and smallmolecules can be distinguished by utilizing arrays of aptamers thattarget different regions of target proteins or small molecules. Hence,the Apta meter technology of the present invention is facilitated byaptamer discovery methods such as those utilized by Orthosystems, Inc.which typically return a number of different aptamers for the sametarget, with different affinities and recognition regions. Theconsequences of this enhanced stringency of the Apta-meter fordiagnostic applications is a significant elimination of false positivein diagnostic applications, e.g. for cardiac or cancer biomarkers. Theability to reduce false-positives for low-cost point-of-care devicesunder situations where rapid therapeutic decisions must be made in turnrepresents a diagnostic tool with wide-ranging public healthimplications.

CSPs are designed using principles of oligonucleotide thermodynamics tocreate a series of metastable probe-competitor pairs that differ instability in the presence of target. A series of CSPs for detecting andquantitating a specific target domain will typically all contain thesame probe (assuring identical hybridization specificity for eachindividual CSP) but different associated competitors which serve tomodulate hybridization affinity through competitive equilibrium with thetarget domain. This results in the differing concentration dependentresponse of the target to each individual CSP even though they all havethe same specificity in target binding.

The DNA meter comprises a series of digital switches that define nucleicacid target identities and absolute concentrations by the types andnumbers of switches of each type that are turned “on”. When applied tothe analysis of PCR amplified products, the DNA meter will provide rapididentification and quantification of DNA targets after a limited numberof cycles. Besides being more rapid than conventional qPCR, the DNAmeter can be used to rapidly localize mutations that would not beapparent with conventional qPCR. For situations where a fixed series oftarget molecules must be repetitively determined, and/or screened formutations, the DNA meter thus provides a rapid, cost-effective andsignificantly more informative alternative to qPCR. In addition toproviding an alternative to qPCR, DNA meters can enhance qPCR by beingdirectly integrated as detection arrays into qPCR machines. In thisembodiment, the technology of the present invention provides a powerfultool for quantitative mutational and gene expression analysis.

In one embodiment, the “target” for the competitive surface probe couldalso be a fluorescently labeled solution probe that binds the analyte(biologically relevant target) in solution. When the analyte is absent,then the solution probe binds as a “target” to the competitive surfaceprobe, and the spot lights up indicating the absence of analyte. In thepresence of analyte, the spots would then turn off, in a concentrationdependent manner. An alternative means of doing the same thing would beto have the “target” for the surface binary probe to be labeled withquencher, and have the binary probe labeled with fluorophore. In thiscase the presence of analyte would remove the surface-bound quencher,causing the spots to light up in a concentration dependent manner. Thisembodiment represents a solution binding but surface monitoring versionof a self-reporting quantitative recognition array.

The invention described herein builds on the use of solution competitiveprobes for rapid thermodynamic screening. However, in contrast to theprior solution work, which is limited by the concentrations required forspectroscopic or thermodynamic measurements, molecular constructs of thepresent invention, such as surface probes, allow measurements to beaccessible over a very wide range of target concentrations. Whenconfigured such that the targets for the CSP probes are themselvesprobes for solution analytes, then the quantitative surface arraysprovide a label-free means of monitoring interactions in solution. DNAmeters comprising arrays of CSPs targeting multiple regions per ampliconmay be integrated into a variety of biosensor detection schemes in orderto facilitate the rapid identification and localization of geneticanomalies. Apta-Meter arrays also allow precise, multicomponent analysisof complex biological samples. Consequently, DNA meter and Apta-Metertechnology of the present invention may be used in a range of research,forensic and diagnostic applications.

Other examples for using the quantitative recognition arrays of theinstant invention include surveillance, diagnosis and genotyping ofantibiotic-resistant bacterial infections (e.g., S. aureus, C.difficile), and neonatal genotyping. The technology of the presentinvention is also uniquely suited for forensic applications. A modelDNA-meter may be designed for its ability to define short tandem repeat(STR) length distributions on-chip, by a hybridization-based assay notinvolving electrophoretic separation. Applications relevant to thetherapeutics include, without limitation, on-line sensors for bioprocessquality control of antibodies and other therapeutic proteins. Thetechnology of the present invention may also be used for rapidmonitoring of air, water and food for potential toxic and/or infectiousagents.

Definitions

“Molecular construct” means a molecular structure, complex, or a partthereof and includes, for example, a probe complex. A “probe complex”may be a molecular construct comprising a target-binding domain.

“Energy landscape” means the points of an energy surface mappingpossible conformations of a molecular structure with their correspondingGibbs free energy levels on a two-, or three-, or n-dimensionalcoordinate system. The coordinate system would include averagedintermolecular spatial coordinates (e.g. x, y, and z) as well ascoordinates relating to thermodynamic ensemble variables (e.g.temperature, pressure, concentration).

“Feature density” means the number of molecular constructs per spatialunit, that is per unit length for a one spatial dimension system, perunit area for a two spatial dimension system, per unit volume for athree spatial dimension system.

“Metastable equilibrium” means a local Gibbs free energy minimum of astate on an energy landscape comprising the initial Gibbs free energyground state of an unhybridized probe and an unbound target (or anunhybridized probe and an unbound off-target, as the case may be) andthe final Gibbs free energy ground state of a probe-target complex (or aprobe-off-target complex, as the case may be), the initial ground statebeing at a higher Gibbs free energy than the final ground state.

As used herein, the term “stable” may refer to a free energy state thatis preferred over alternative free energy states under a defined set ofconditions, including, e.g., temperative, ionic conditions and thepresence or absence of substances, molecules or environmental factorsthat influence molecular or intermolecular conformation. Stability doesnot imply indefinite irreversibility, but may be used in reference tovarious states of relative irreversibility, quasi-irreversibility,pseudo-irreversibility, quasi-reversibility or pseudo-reversibility asused in the art. Quasi-irreversible states include, for example,“metastable states” as defined herein.

The phrase “solid support” refers to any solid surface including,without limitation, any chip (for example, silica-based, glass, or goldchip), glass slide, membrane, plate, bead, solid particle (for example,agarose, sepharose, polystyrene or magnetic bead), column (or columnmaterial), test tube, or microtiter dish/plate. Solid support may bemade out of, without limitation, nitrocellulose, glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, gabbros, and magnetite.

The term “isolated” may refer to a compound or complex that has beensufficiently separated from other compounds with which it wouldnaturally be associated. “Isolated” is not meant to exclude artificialor synthetic mixtures with other compounds or materials, or the presenceof impurities that do not interfere with fundamental activity or ensuingassays, and that may be present, for example, due to incompletepurification, or the addition of stabilizers.

The term “DNA-meter” as used herein contemplates not only meters basedon DNA and alternatives stated herein, but is also intended to refer tometers based on DNA analogs, RNA, RNA analogs and, more generally,nucleic acids and nucleotide-based molecular mimics, where molecularmimics are natural or synthetic nucleotide molecules or groups ofmolecules designed, selected, manufactured, modified or engineered tohave a structure or function equivalent or similar to the structure orfunction of a different nucleotide molecule or group of molecules.Examples of such DNA and RNA analogs include but are not limited tobackbone modifications (e.g. locked nucleic acids, peptide nucleicacids), base modifications, xeno-nucleic acids (Pinheiro et al. (Science(2012) 336:341-344), abasic sites and unnatural base analogs.

The term “Apta-meter” as used herein contemplates not only to metersbased on nucleic acid aptamers and alternatives stated herein, but isalso intended to refer to meters based on nucleic acid analogs,including backbone modifications (e.g. locked nucleic acids, proteinnucleic acids) base modifications, abasic sites, unnatural base analogsand synthetic non-nucleic acid molecules with structural and recognitionfeatures designed to mimic known aptamers. Aptamers includesingle-stranded, partially single-stranded, partially double-stranded ordouble-stranded nucleotide sequences capable of specifically recognizinga selected nonoligonucleotide molecule or group of molecules by amechanism other than Watson-Crick base pairing or triplex formation.Aptamers disclosed herein include, without limitation sequencescomprising nucleotides, ribonucleotides, deoxyribonucleotides,nucleotide analogs, modified nucleotides and nucleotides comprisingbackbone modifications, branchpoints and nonnucleotide residues, groupsor bridges. They may be partially and fully single-stranded anddouble-stranded nucleotide molecules and sequences, synthetic RNA, DNAand chimeric nucleotides, hybrids, duplexes, heteroduplexes and anycomplex, conjugate or nucleotide-base molecular mimic thereof.

As used herein, a “biological sample” refers to a sample of biologicalmaterial obtained from a subject, particularly a human subject,including a tissue, a tissue sample, a cell sample, a tumor sample, anda biological fluid (e.g., blood or urine).

The term “probe” as used herein refers to an oligonucleotide,polynucleotide, nucleic acid, either RNA or DNA, or other hybridizableentity (e.g. PNAs, locked nucleic acids, or other base or backbonemodifications), whether occurring naturally as in a purified restrictionenzyme digest or produced synthetically, which is either a) capable ofannealing with or specifically hybridizing to a nucleic acid withsequences complementary to the probe or b) capable of binding with highaffinity to a non-nucleic acid target. A probe may be single-stranded,double-stranded, or multistranded. The exact length of the probe willdepend upon many factors, including temperature, source of probe and useof the method. For example, for diagnostic applications, depending onthe complexity of the target sequence, the oligonucleotide probetypically contains about 10-100, about 10-50, about 15-30, about 15-25,about 20-50, or more nucleotides, although it may contain fewernucleotides. The probes herein may be selected to be complementary todifferent strands of a particular target nucleic acid sequence. Thismeans that the probes must be sufficiently complementary so as to beable to “specifically hybridize” or anneal with their respective targetstrands under a set of pre-determined conditions. Therefore, the probesequence need not reflect the exact complementary sequence of thetarget, although they may. For example, a non-complementary nucleotidefragment may be attached to the 5′ or 3′ end of the probe, with theremainder of the probe sequence being complementary to the targetstrand. Alternatively, non-complementary bases or longer sequences canbe interspersed into the probe, provided that the probe sequence hassufficient complementarity with the sequence of the target nucleic acidto anneal therewith specifically.

With respect to single stranded nucleic acids, particularlyoligonucleotides, the term “specifically hybridizing” refers to theassociation between two single-stranded nucleotide molecules ofsufficiently complementary sequence to permit such hybridization underpre-determined conditions generally used in the art (sometimes termed“substantially complementary”). In particular, the term refers tohybridization of an oligonucleotide with a substantially complementarysequence contained within a single-stranded DNA molecule of theinvention, to the substantial exclusion of hybridization of theoligonucleotide with single-stranded nucleic acids of non-complementarysequence. Appropriate conditions enabling specific hybridization ofsingle stranded nucleic acid molecules of varying complementarity arewell known in the art.

The term “oligonucleotide” as used herein refers to nucleic acids,sequences, primers and probes of the present invention, and is definedas a nucleic acid molecule comprised of two or more ribo- ordeoxyribonucleotides (or analogs thereof), preferably more than three.The exact size of the oligonucleotide will depend on various factors andon the particular application and use of the oligonucleotide.

“Nucleic acid” or a “nucleic acid molecule” as used herein refers to anyDNA or RNA molecule, single, double, or multi stranded and, if singlestranded, the molecule of its complementary sequence in either linear orcircular form. Nucleic acid molecules of the instant invention may beoligonucleotides or synthetic polynucleotides and selected nucleic acidsequences which may optionally be conjugated to one or morenonoligonucleotide molecules. Nucleic acid molecules of the instantinvention may comprise one or more modifications such as backbone and/orbase modifications/analogs (e.g., LNAs) or xeno-nucleic acids. Indiscussing nucleic acid molecules, a sequence or structure of aparticular nucleic acid molecule may be described herein according tothe normal convention of providing the sequence in the 5′ to 3′direction. With reference to nucleic acids of the invention, the term“isolated nucleic acid” is sometimes used. This term, when applied toDNA, refers to a DNA molecule that is separated from sequences withwhich it is immediately contiguous in the naturally occurring genome ofthe organism in which it originated. For example, an “isolated nucleicacid” may comprise a DNA molecule inserted into a vector, such as aplasmid or virus vector, or integrated into the genomic DNA of aprokaryotic or eukaryotic cell or host organism.

The term “nucleotide” includes nucleotides and nucleotide analogs,preferably groups of nucleotides comprising oligonucleotides, e.g., anycompound containing a heterocyclic compound bound to a phosphorylatedsugar by an N-glycosyl link or any monomer capable of complementary basepairing or any polymer capable of hybridizing to an oligonucleotide.

The term “nucleotide analog” refers to molecules that can be used inplace of naturally occurring bases in nucleic acid synthesis andprocessing, preferably enzymatic as well as chemical synthesis andprocessing, particularly modified nucleotides capable of base pairingand optionally synthetic bases that do not comprise adenine, guanine,cytosine, thymidine, uracil or minor bases. This term includes, but isnot limited to, modified purines and pyrimidines, minor bases,convertible nucleosides, structural analogs of purines and pyrimidines,labeled, derivatized and modified nucleosides and nucleotides,conjugated nucleosides and nucleotides, sequence modifiers, terminusmodifiers, spacer modifiers, and nucleotides with backbonemodifications, including, but not limited to, ribose-modifiednucleotides, phosphoramidates, phosphorothioates, phosphonamidites,methyl phosphonates, methyl phosphoramidites, methyl phosphonamidites,5′43-cyanoethyl phosphoramidites, methylenephosphonates,phosphorodithioates, peptide nucleic acids, achiral and neutralintemucleotidic linkages and nonnucleotide bridges such as polyethyleneglycol, aromatic polyamides and lipids.

When applied to RNA, the term “isolated nucleic acid” refers primarilyto an RNA molecule encoded by an isolated DNA molecule as defined above.Alternatively, the term may refer to an RNA molecule that has beensufficiently separated from other nucleic acids with which it would beassociated in its natural state (i.e., in cells or tissues). An“isolated nucleic acid” (either DNA or RNA) may further represent amolecule produced directly by biological or synthetic means andseparated from other components present during its production.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight of a given material (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-95% by weightof the given compound. Purity is measured by methods appropriate for thegiven compound (e.g., chromatographic methods, agarose or polyacrylamidegel electrophoresis, HPLC analysis, and the like).

The term “isolated protein” or “isolated and purified protein” issometimes used herein. This term refers primarily to a protein producedby expression of an isolated nucleic acid molecule of the invention.Alternatively, this term may refer to a protein that has beensufficiently separated from other proteins with which it would naturallybe associated, so as to exist in “substantially pure” form. “Isolated”is not meant to exclude artificial or synthetic mixtures with othercompounds or materials, or the presence of impurities that do notinterfere with the fundamental activity, and that may be present, forexample, due to incomplete purification, or the addition of stabilizers.

As used herein, the term “array” refers to an ordered arrangement ofhybridizable array elements (e.g., polypeptides, proteins, nucleicacids, antibodies, small molecules, etc.). The array elements arearranged so that there are at least one or more different array elementson a solid support. The array elements may be arranged in one or moredimensions. In a particular embodiment, the array elements compriseoligonucleotide probes.

As used herein, a “specific binding pair” comprises a specific bindingmember and a binding partner which have a particular specificity foreach other and which in normal conditions bind to each other inpreference to other molecules. Examples of specific binding pairsinclude, without limitation, antigen-antibody, receptor-hormone,receptor-ligand, agonist-antagonist, lectin-carbohydrate, nucleic acid(e.g., RNA or DNA) hybridizing sequences, nucleic acid-protein, andpolypeptide-small molecule. Various other specific binding pairs arecontemplated for use in practicing the methods of this invention, suchas will be apparent to those skilled in the art.

The following non-limiting examples are provided to further illustratethe present invention.

Example 1

A DNA-meter was constructed which can distinguish S. aureus VanA targetfrom MecA target. The DNA-meter also shows a concentration-dependentresponse over a wide concentration range. Specifically, a set of fiveCSPs was designed (Table I). Table 1 provides the T_(1/2) concentrationsat 30° C. These CSPs were used to derivatize dextran-coated slides withspots. The slide yielded a concentration-dependent capture of target andwas able to discriminate between target mecA from off-target vanA. Itwas also demonstrated that vanA target lights up spots derivatized witha surface bound vanA probe, but not spots derivatized with any of themecA probes. The lower limit of target monitored was 100 fM and theupper limit was 100 nM. The results of these experiments are summarizedin Table II.

TABLE I CSPs used to derivatize dextran. Probe Probe StrandCompetitor Strand name (SEQ ID NO) (SEQ ID NO) (T_(1/2))calc MecPQT1TATGTATGCT none 5 x 10⁻¹⁸ TTGGTCTTTC TG-NH₂ (1) M VanP0 NH₂-GTGAGGTCGGnone 1 X 10⁻¹⁸ TTGTGCGGTA TTGGG (2) M MecPQ3 TATGTATGCT NH₂-CAGAAAGACT138 fM TTGGTCTTTC TG-NH₂ (1) TTTTTTTTGC ATACATA (3) MecPQ8TATGTATGCT TTGGTCTTT NH₂-CAGAAAGACC  17 pM CTG-NH₂ (1) TTGCATACAT A (4)MecPQ14 TATGTATGCT TTGGTCTTT NH₂-CAGAAAGACC  11 μM CTG-NH₂ (1)AAAGCATACA TA (5)

TABLE II Probe Fluorescence for Various Target Concentrations. MecA VanAVan target target P0 MecPQT1 MecPQ3 MecPQ8 MecPQ14 100 fM 0 − + − − − 1pM 0 − + − − − 10 pM 0 − + + − − 100 pM 0 − + + + − 10 nM 0 − + + + −100 nM 0 − + + + − 0   100 nM + − − − −

In performing these initial experiments, polytetrafluoroethylene(PTFE)-coated glass slides with 4 mm diameter wells (SPI supplies, WestChester, Pa.) were used. The glass wells were uniformly coated withdextran, as described above. The use of PTFE-coated slides allowed forthe use of the cover-slips to maintain a solution environment over theslides during the measurements and allowed for the complete immersion ofthe slides in target solutions. Into each well square arrays of 4 spotswere manually spotted, using 0.3 μl of 10 μM solutions of amino-labeledprobe. The volume of the pipeting was controlled by using a NanoFil™syringe (World Precision Instruments, Sarasota, Fla.) that was modifiedto convert a defined turn of a screw to a defined linear distance of thesyringe cylinder and, thus, to a reasonably well defined nanolitervolume of solution. Spots were pipetted onto the NaIO₄ activated dextrancoated surfaces in the presence of NaCNBH₃. The resultant spots haddiameters in the range of 200-400 μm and were well separated from eachother. Subsequent reaction with NaBH₄ reduced any remaining aldehydes tohydroxyls. All spotting was performed at least in quadruplicate and eacharray of 4 spots had at least one control spot that was derivatized witha 10mer directly labeled with Cy3. The presence of this spot provided uswith the ability to accurately locate the other three spots, whichcorresponded to various combinations of the other probes listed inTables I and II.

For the lower target concentrations tested, spotted slides werecompletely submerged in 50 ml centrifuge tubes containing targetdissolved in 1×SSC buffer (150 mM NaCl, 15 mM sodium citrate, pH 7). Inorder to address potential hysteresis effects tubes containingmicroscope slides were heated to 90° C. and allowed to cool toroom-temperature before removing slides, covering with target solutionfrom the tube, and a cover-slip, and taking measurements.

In the presence of 100 fM through 1 pM of Cy3 labeled mecA target, thecontrol spots and the spots derivatized with MecPQT1 probe lit up withfluorescence intensity in the Cy3 channel, but none of the other spotsshowed discernable signal. In the presence of 10 pM total concentrationof mecA target the mecPQ3 probe spots also light up. For 100 pM mecAtarget, fluorescence is clearly observable for the mecPQ8 spots as well.For 100 fM through 100 nM mecA target, no fluorescence is apparent foreither the vanA P0 probe spots or the mecPQ14 probe spots. When surfacefluorescence for a target containing 1 μM labeled mecA probe wasmonitored, the background fluorescence of the solution prohibitedmonitoring surface fluorescence. Finally, in the presence of van Atarget at 100 nM total concentration, only the control spots and thevanA P0 probe spots show fluorescence when monitored using the Cy3filter cube.

Representative spots are shown in FIG. 2. These spots were covered withMecA target solution at the concentrations indicated and imaged througha cover-slip. The exposure time for all images was constant at threeseconds. Data were collected using a black and white camera andcolorized using ImageJ software so that the most intense fluorescencewas represented by white, followed by yellow, followed by green,followed by black.

Example 2

The basic features of the behavior of CxP probes, either in solution orattached to surfaces (CSPs), as for the DNA Meter and the Apta-Meter,reflect a very simple model:

P_(C)↔P_(H) and P_(C)+T↔P_(T), which are governed by the equilibriumexpressions:

K₁=(P_(H))/(P_(C)) and K_(lin)=(PT)/(P_(C))(T), where P_(C) and P_(H)are, respectively, the coil and partially hybridized forms of the CxPprobe (i.e., before target binding), and where PT is the probe-targetcomplex. Making use of mass balance, these equations are easily solvedto give f_(B), the fraction of probe molecules that are bound by target.By setting f_(B)=½, an equation is obtained for the midpoint targetconcentration for ½ saturation of probe: (T)_(1/2)=(1+K₁)/K_(lin). Fromthis equation, it is apparent that as K₁ increases, the criticalconcentration shifts to higher total concentration. For solutionmeasurements, a more relevant equation expresses the total concentrationof target at half-saturation:

(T_(tot))_(1/2)=(1+K₁)/K_(lin)+(P_(tot)), where (P_(tot)) is the totalconcentration of probe in solution.

It is apparent that, in this case, half saturation does not typicallyoccur until the target concentration approaches that of the probe.Simulations using this formalism are shown in FIG. 4. Two differentsituations are considered. For FIG. 4A, (T_(tot)) is taken to be inconsiderable excess compared to (P_(tot)), as would be the case forprobes attached to biosensor surfaces. In FIG. 4B, (P_(tot)) is fixed at10 nM, a typical value for solution fluorescence measurements. Forcomparison, FIG. 4C shows curves for the binding of the complementarycoil probe to target as a function of temperature, again underconditions of excess target. Clearly, such temperature curves showsignificant similarity to the results of FIG. 4A, supporting theposition that stringency can be enhanced by reducing affinity, in asimilar manner using either temperature or competitive interactions.

In performing these simulations, competitive hybridization probes weredesigned based on a 30-base pair recognition sequence from the mecA gene(Sinsimer et al. (2005) J. Clin. Microbiol., 43:4585-91). This targetsequence is given by

(SEQ ID NO: 6) TGC AGA AAG ACC AAA GCA TAC ATA TTG AAA.The constant recognition sequence for this target is indicated byunderlining. As probes, the sequence TT CAA TAT GTA TGC TTT GGT CTT TCTG (SEQ ID NO: 7) was used for strand P. Thermodynamic parameters for theformation of duplex from the target sequence binding to linear probe at0.225 M NaCl are calculated using nearest neighbor methods (Markham etal. (2005) Nucl. Acids Res., 33:W577-81). The solution monomolecularconstructs for which these simulations are made are shown in Table III,together with the calculated concentrations for half-saturation,(T)_(1/2).

TABLE III Ligands for simulations, with calculated (T)_(1/2) at 30° C.Probe Sequence (SEQ ID NO) (T)_(1/2) PQT1 TATGTATGCTTTGGTCTTTCTG (1)  5 aM Mec TATGTATGCTTTGGTCTTTCTG (1) 138 fM PQ3CAGAAAGACTTTTTTTTTGCATACAT A (3) Mec TATGTATGCTTTGGTCTTTCTG (1)  17 pMPQ8 CAGAAAGACCTTGCATACATA (4)

Even though these calculations are performed assuming that the target isa nucleic acid strand that hybridizes to the probe sequence, exactly thesame formalism applies for binding of a protein to an aptamer sequencethat is in equilibrium with a partially complementary competitivestrand. For the aptamer case, the ability to fine-tune stringency viacompetitive interactions is particularly important since denaturationand aggregation can significantly limit the temperature range over whichprotein-ligand interactions can be interrogated.

Example 3

Experiments can be performed to monitor PCR in real-time chambersderivatized with CSP probes for S. aureus genes. The primers will be asdescribed above for the S. aureus target genes (Sinsimer et al. (2005)J. Clin. Microbiol., 43:4585-91). S. aureus and other bacterial DNAsfrom normal and methicillin resistant strains will be obtained fromATCC. Bacterial DNA was sealed with buffer and enzymes in the chamber.PCR was performed using previously described protocols (Sinsimer et al.(2005) J. Clin. Microbiol., 43:4585-91). Chambers modified with CSPswere used to monitor the amplification of target sequences representingthe SG16S, BAC16S, spa and mecA genes (Table IV). Followingamplification, the amplified DNA was collected and subjected togel-electrophoresis.

TABLE IV Linear probes for S. aureus PCR. GeneLinear probe sequence (SEQ ID NO) BAC16S5′SH-(CH₂)₆-CGAGCTGACGACARCCATGCA3′ (8) SG16S5′SH-(CH₂)₆-CTTACCAAATCTTGACATCCT3′ (9) spa5′SH-(CH₂)₆-TTGTTGAGCTTCATCGTGTTG 3′ (10) mecA5′SH-(CH₂)₆-TTCAATATGTATGCTTTGGTCTTTCTG3′ (7)

Preliminary results with the mecA gene have shown that having surfacebound probes enhances sensitivity and thus reduces the number of cyclesrequired to obtain a quantitative result. In comparison to using gelelectrophoresis for analysis and quantification, the use of CSPs reducedby 75% the number of cycles required to obtain enough DNA for analysis.Moreover, using multiple probes for the same target sequences andmultiple target sequences per amplicon both enhances quantitativeaccuracy and reduces the occurrence of false positives. Mostsignificantly, the use of a DNA meter integrated into a quantitative PCRdevice will dramatically enhance the ability to discriminate SNPs,lesions and other genetic defects. In this application, series of CSPscan be designed to target multiple regions along individual amplicons.In the absence of mutations or other genetic anomalies, the order ofturning on of CSPs for the particular sequences will show acharacteristic pattern. In the presence of mutations, this order ofturning on will deviate for those sequences where genetic anomaliesoccur. An illustration of this ability to distinguish genetic anomaliesis shown in FIG. 5. In these calculations a competitive surface probefor the mecA gene from S. aureus was used and thermodynamic parameterswere estimated from nearest neighbor calculations. The effect ofcompetition for the target from the complement strand, which is assumedto be amplified at the same rate as the target strand, was included,thereby making the equations somewhat more complex than presented above.In FIG. 5A are calculations assuming a hypothetical solution molecularbeacon probe with identical thermodynamic parameters as the CSP. Forboth simulations, an initial target concentration of 10⁻¹⁵ M and anefficiency of 1 is assumed. The molecular beacon concentration is takenas 100 nM, which is a value within the typical range used for molecularbeacons in solution. From this curve a result is illustrated that iswell-appreciated by practitioners of the molecular beacon art: It isimpossible to distinguish closely related targets based on cycle numberalone. In contrast, as is shown in FIG. 5B, when measurements can beperformed for surface bound probes, where the concentration of target isin significant excess over the concentration of probe, then the cyclenumber is determined by the dissociation constant for formation ofprobe-target complex, and targets differing by as little as a singlemismatch are readily distinguishable. As such, inexpensive electronicqPCR devices are developed for rapid, specific and highly sensitivedetection of unlabeled nucleic acid targets.

Example 4

In one manifestation of the “tuning fork” probe concept describedherein, the availability of the “free” probe strand (P) forbinding/hybridizing to its complementary target domain (T) issystematically and predictably modulated. This modulation is achieved bypre-associating the probe strand with a family of competitor strands (C)of identical Watson Crick sequence recognition elements that arecomplementary to the probe, but which bind the probe with varyingstrengths due to incorporation into the C strand of“recognition-neutral” T bulge loops of increasing size, thereby creatinga ladder of energetically tuned competitor strands (C*). Thecompetitor-probe complex (C*P) becomes incrementally less stable withincreasing bulge T-loop size, causing the competitor (“masking tape”)strand (C*) to be more readily displaced at lower concentrations oftarget (T), thereby resulting in the formation of the final probe-targetcomplex (PT); an event which corresponds to a successful “hit.” FIG. 6provides a schematic representation.

This design concept can be reduced to practice by either decorating asurface with or making a solution mixture of a family of energeticallytuned C*P constructs (“tuning forks”), thereby forming an energeticladder of probe complexes (C*P) against which one can titrate Targetsamples of increasing concentrations. In a successfully designed system,at the lowest target concentrations, “hits” (formation of the PTcomplex) will be detected initially by displacement of the leaststrongly bound “masking tape” C* strand. With increasing Tconcentrations, subsequent hits will sequentially be detected by andscale with the increasing stability of the C*P family of tuning forkcomplexes.

Here, spectroscopic and calorimetric techniques were used to demonstrateproof of principle for this methodology. It is shown in solution thatenergetically tuned probe complexes (C*P) not only can selectivelydetect the presence of target sequences, but also can quantitate theamount of target present. Specifically, a strand displacement reactionwas conducted and monitored by optical methods using the all-T bulgeloops (composed of a strand with variable length T loops and a 22mer) asCompetitor probe complex and the complementary 22mer as single strandtarget (see FIG. 6). Notably, the temperature at which the competitorbecomes displaced by the Target strand decreases with decreasing thermalstability of the probe competitor complex, i.e. T8 is displaced prior toT4 and T2 (see FIG. 7). Thus, the more the probe competitor complex isdestabilized by increasing the loop size in the competitor strand, thelower the temperature at which strand exchange occurs. In other words,the system can be tuned in a way that yields the resolution andspecificity desired. The thermal stability of the resulting probe-targetcomplex is not affected by the presence or nature of the competitor (C*)strand.

Additional optical and calorimetric data shown in FIG. 8 furtherdemonstrate proof of principle of this next generation methodology. Thecrucial observation is the sequential disappearance of the lowtemperature segment of the composite T2●P, T4●P, and T8●P melting curveand the simultaneous appearance and increase in the P●T meltingtransition with increasing Target T concentration. As Target strandconcentration increases, the thermally least stable component of theT2●P, T4●P, and T8●P mixture (T8●P, first then T4●P) undergoes strandexchange and consequently disappears from the composite melting curve,while the more stable components (primarily T2●P) remains unchanged.

Several publications and patent documents are cited throughout thespecification in order to describe aspects of the present invention.Each of these references is incorporated herein as though set forth infull.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

What is claimed is:
 1. A molecular construct for differentiating atarget molecule from an off-target molecule, said target molecule havinga target domain and said off-target molecule having an off-target domainthat differs from said target domain, said molecular constructcomprising a) a first domain that is capable of binding to said targetdomain, and b) a second domain that is at least partially hybridizablewith said first domain, wherein said first domain is capable of bindingto said off-target domain; wherein a hybrid of said first domain withsaid second domain is in a state of predetermined stable equilibrium inthe absence of said target domain and in a state of predeterminedmetastable equilibrium in the presence of said target domain; andwherein the free energy of displacement of said second domain by saidtarget domain from said hybrid of said first domain with said seconddomain is energetically more favored than the free energy ofdisplacement of said second domain by said off-target domain from saidhybrid of said first domain with said second domain.
 2. The molecularconstruct of claim 1, wherein said off-target domain is generallyhomologous with respect to said target domain.
 3. The molecularconstruct of claim 1, wherein said off-target domain differs from saidtarget domain either chemically by at least one functional group orconformationally or both.
 4. A population of molecular constructs fordifferentiating a target molecule from an off-target molecule, saidtarget molecule having a target domain and said off-target moleculehaving an off-target domain that differs from said target domain, eachmolecular construct comprising a) a first domain that is capable ofbinding to said target domain, and b) a second domain that is at leastpartially hybridizable to said first domain, wherein said first domainis capable of binding to said off-target domain; wherein a hybrid ofsaid first domain with said second domain is in a state of predeterminedstable equilibrium in the absence of said target domain and in a stateof predetermined metastable equilibrium in the presence of said targetdomain; and wherein the free energy of displacement of said seconddomain by said target domain from said hybrid of said first domain withsaid second domain is energetically more favored than the free energy ofdisplacement of said second domain by said off-target domain from saidhybrid of said first domain with said second domain.
 5. The populationof claim 4, wherein said off-target domain is generally homologous withrespect to said target domain.
 6. The population of claim 4, whereinsaid off-target domain differs from said target domain either chemicallyby at least one functional group or conformationally or both.
 7. Thepopulation of claim 4, wherein said population comprises: a) a firstsubpopulation of at least one said molecular construct with respect to afirst target domain, and b) a second subpopulation of at least one saidmolecular construct with respect to a second target domain, wherein saidfirst target domain differs from said second target domain.
 8. Thepopulation of claim 7, wherein said first target domain and said secondtarget domain are disposed on the same target molecule.
 9. Thepopulation of claim 7, wherein said first target domain and said secondtarget domain are disposed on different target molecules.
 10. Thepopulation of claim 7, wherein said first target domain is an off-targetdomain with respect to a second target molecule.
 11. The population ofclaim 4, wherein said population comprises: a) a first subpopulationcomprising at least one said molecular construct with respect to a firstoff-target domain and b) a second subpopulation comprising at least onesaid molecular construct with respect to a second off-target domain, andwherein said first off-target domain differs from said second off-targetdomain.
 12. The population of claim 11, wherein said first off-targetdomain and said second off-target domain are disposed on the sameoff-target molecule.
 13. The population of claim 11, wherein said firstoff-target domain and said second off-target domain are disposed ondifferent off-target molecules.
 14. The population of claim 11, whereinsaid first off-target domain is a target domain with respect to a secondoff-target molecule.
 15. The population of claim 4, wherein a) thesecond domain of said molecular constructs of a first portion of saidpopulation comprises a first subdomain that is at least partiallycomplementary to the first domain and a second subdomain that is notcomplementary to the first domain; and b) the second domain of saidmolecular constructs of a second portion of said population comprises afirst subdomain that is at least partially complementary to the firstdomain and a second subdomain that is not complementary to the firstdomain; wherein the second subdomain of the molecular constructs of thefirst portion differs from the second subdomain of the molecularconstructs of the second portion and modulates the binding of the firstdomains to the second domains of the first portion with respect to thebinding of the first domains to the second domains of the secondportions.
 16. An array comprising more than one biosensor element on asolid support, wherein each biosensor element comprises a molecularconstruct in accordance with claim
 1. 17. A kit comprising at least onemolecular construct of claim
 1. 18. A method for differentiating atarget molecule from an off-target molecule, said method comprisingcontacting a molecular construct with an unknown molecule.
 19. A methodfor detecting the presence of a nucleic acid sequence of interest, saidmethod comprising: a) contacting a population of probe complexes with asample, wherein said population of probe complexes comprises at least i)a first subpopulation of probe complexes comprising a probe strand and acompetitor strand, wherein said competitor strand comprises at least onehybridization domain and at least one domain which does not hybridizewith said probe strand, wherein said hybridization domain is at leastpartially complementary to said probe strand, and ii) a secondsubpopulation of probe complexes comprising a probe strand and acompetitor strand, wherein said competitor strand comprises at least onehybridization domain and at least one domain which does not hybridizewith said probe strand, wherein said hybridization domain is at leastpartially complementary of said probe strand, wherein thenon-hybridization domain of the second subpopulation differs from thenon-hybridization domain of the first subpopulation; and b) detectingthe formation of complexes between the probe strand and a nucleic acidmolecule from the sample, wherein the presence of such complexes isindicative of the presence of the nucleic acid sequence of interest. 20.The method of claim 19, wherein said competitor strand comprises twohybridization domains separated by a domain which does not hybridizewith said probe strand.
 21. The method of claim 19, wherein saidhybridization domains are the complement of said probe strand.
 22. Themethod of claim 19, wherein the nucleic acid molecules of the sample aredetectably labeled.
 23. The method of claim 19, wherein said populationof probe complexes are in solution.
 24. The method of claim 19, whereinsaid population of probe complexes is spatially constrained.
 25. Themethod of claim 22, wherein said population of probe complexes is linkedto a solid support.
 26. The method of claim 19, wherein step a) isperformed with increasing concentrations of sample nucleic acidmolecules.
 27. The method of claim 19, wherein the non-hybridizationdomain comprises a loop domain.
 28. An array of a plurality ofpopulations in accordance with claim 4 for detecting a plurality ofdifferent target molecules in a sample, wherein the free energy ofdisplacement of each of those populations differs with respect to eachother of those populations so as to be capable of identifying differenttarget molecules in said sample.
 29. The array of claim 28, wherein thenumber of different targets is from about 2 to
 10. 30. The array ofclaim 28, wherein the number of different targets is from 100 to 1000.31. The array of claim 28, wherein the number of different targets isgreater than
 1000. 32. The array of claim 28, wherein the ratio of thefree energy of displacement of one population to another population isabout 1.1.
 33. The array of claim 28, wherein the ratio of the freeenergy of displacement of one population to another population is from1.1 to 1.2.
 34. The array of claim 28, wherein the ratio of the freeenergy of displacement of one population to another population is from1.2 to 1.5.
 35. The array of claim 28, wherein the ratio of the freeenergy of displacement of one population to another population is from1.5 to 2.0.
 36. The array of claim 28, wherein the ratio of the freeenergy of displacement of one population to another population isgreater than
 2. 37. An array of a plurality of populations in accordancewith claim 4 for determining the relative number of target molecules ina sample, wherein the free energy of displacement of each of thosepopulations differs with respect to each other of those populations soas to be capable of determining the relative number of target moleculesof a population from another population in said sample.
 38. The array ofclaim 37, wherein the number of different targets is from about 2 to 10.39. The array of claim 37, wherein the number of different targets isfrom 100 to
 1000. 40. The array of claim 37, wherein the number ofdifferent targets is greater than
 1000. 41. The array of claim 37,wherein the ratio of the free energy of displacement of one populationto another population is about 1.1.
 42. The array of claim 37, whereinthe ratio of the free energy of displacement of one population toanother population is from 1.1 to 1.2.
 43. The array of claim 37,wherein the ratio of the free energy of displacement of one populationto another population is from 1.2 to 1.5.
 44. The array of claim 37,wherein the ratio of the free energy of displacement of one populationto another population is from 1.5 to 2.0.
 45. The array of claim 37,wherein the ratio of the free energy of displacement of one populationto another population is greater than
 2. 46. An array of a plurality ofpopulations in accordance with claim 4 for identifying a plurality ofdifferent target molecules in a sample and determining the relativenumber of each of those different target molecules in the sample,wherein a) the free energy of displacement of each of those populationsdiffers with respect to each other of those populations so as to becapable of identifying each different target molecule in said sample andb) the free energy of displacement of each of those populations differswith respect to each other of those populations so as to be capable ofdetermining the relative number of each of those different targetmolecules of a population from another population in said sample. 47.The array of claim 46, wherein the number of different targets is fromabout 2 to
 10. 48. The array of claim 46, wherein the number ofdifferent targets is from 100 to
 1000. 49. The array of claim 46,wherein the number of different targets is greater than
 1000. 50. Thearray of claim 46, wherein the ratio of the free energy of displacementof one population to another population is about 1.1.
 51. The array ofclaim 46, wherein the ratio of the free energy of displacement of onepopulation to another population is from 1.1 to 1.2.
 52. The array ofclaim 46, wherein the ratio of the free energy of displacement of onepopulation to another population is from 1.2 to 1.5.
 53. The array ofclaim 46, wherein the ratio of the free energy of displacement of onepopulation to another population is from 1.5 to 2.0.
 54. The array ofclaim 46, wherein the ratio of the free energy of displacement of onepopulation to another population is greater than
 2. 55. A method fordetecting the presence of a nucleic acid sequence of interest, saidmethod comprising: a) contacting a population of molecular constructswith a sample may comprise said nucleic acid sequence, wherein saidpopulation of molecular constructs comprises at least i) a firstsubpopulation of molecular constructs, each molecular constructcomprising a probe strand and a competitor strand, wherein said probestrand comprises at least one hybridization domain and at least oneother domain which does not hybridize with said competitor strand,wherein said at least one hybridization domain is at least partiallycomplementary to said competitor strand, and ii) a second subpopulationof molecular constructs, each molecular construct comprising a probestrand and a competitor strand, wherein said probe strand comprises atleast one hybridization domain and at least one other domain which doesnot hybridize with said competitor strand, wherein said at least onehybridization domain is at least partially complementary to saidcompetitor strand, wherein the non-hybridization domain of the secondsubpopulation differs from the non-hybridization domain of the firstsubpopulation; and b) detecting the formation of complexes between theprobe strand and a nucleic acid sequence from the sample, wherein thepresence of such complexes is indicative of the presence of the nucleicacid sequence of interest.